Ozone and Temperature May Hinder Adaptive Capacity of Mediterranean Perennial Grasses to Future Global Change Scenarios

Climate warming is recognized as a factor that threatens plant species in Mediterranean mountains. Tropospheric ozone (O3) should also be considered as another relevant stress factor for these ecosystems since current levels chronically exceed thresholds for plant protection in these areas. The main aim of the present study was to study the sensitivity of four Mediterranean perennial grasses to O3 and temperature based on plant growth, gas exchange parameters (photosynthesis—A, stomatal conductance—gs, and water use efficiency—WUE), and foliar macro- (N, K, Ca, Mg, P, and S) and micronutrients (B, Cu, Fe, Mn, Mo, and Zn) content. The selected species were grasses inhabiting different Mediterranean habitats from mountain-top to semi-arid grasslands. Plants were exposed to four O3 treatments in Open-Top chambers, ranging from preindustrial to above ambient levels, representing predicted future levels. Chamber-less plots were considered to study the effect of temperature increase. Despite the general tolerance of the grasses to O3 and temperature in terms of biomass growth, WUE and foliar nutrient composition were the most affected parameters. The grass species studied showed some degree of similarity in their response to temperature, more related with phylogeny than to their tolerance to drought. In some species, O3 or temperature stress resulted in low A or WUE, which can potentially hinder plant tolerance to climate change. The relationship between O3 and temperature effects on foliar nutrient composition and plant responses in terms of vegetative growth, A, gs, and WUE constitute a complex web of interactions that merits further study. In conclusion, both O3 and temperature might be modifying the adaptation capacity of Mediterranean perennial grass species to the global change. Air pollution should be considered among the driving favors of biodiversity changes in Mediterranean grassland habitats.


Introduction
Tropospheric ozone (O 3 ) is one of the most relevant air contaminants due to its high toxicity, wide distribution, and its greenhouse effect [1][2][3][4]. Transportation and industrial activity are the main anthropogenic sources of O 3 precursors. However, these pollutants are transported by air masses and can react with precursor emissions from natural sources causing large O 3 surface levels in rural and forested areas far away from anthropic activities [5,6].
The photochemical formation and persistence of O 3 is favored under Mediterranean climatic conditions, causing an important air pollution problem in the Mediterranean Basin [4,[6][7][8]. The current O 3 levels in the Iberian Peninsula chronically exceed the threshold levels for plant protection established by the UNECE Air Convention [9] and the EU Air Quality Directive (2008/50/EC) [10]. This problem is even more acute in the Mediterranean mountains [11,12].  Table 2. Mean meteorological conditions during the experimental assay for the 8-h daylight period 7:00-15:00 (GMT) inside the OTCs and in the AMB plots. Mean and maximum air temperature (mean T, max T), air relative humidity (RH), photosynthetic active radiation (PAR) and vapor pressure deficit (VPD, kPa).

Gas Exchange
Regarding gs ( Figure 2B), three of the four species presented a similar concave tre in response to O3, with a greater stomatal opening under the lowest and highest concentrations (CFA and NFA++ treatments). In A. castellana, NFA and NFA+ treatme were marginally lower than CFA and NFA++ (t20 = 1.79 p = 0.09); but even though tenacissima and F. indigesta showed the same concave pattern, they were not significant = 1.42, p = 0.17; t16 = 1.52, p = 0.15, respectively, for a QE). Contrary to conductance respo in the other species, a marginally CE was observed in F. iberica (t26 = −1.75, p = 0.09).
Consistently with gs values, the most observed WUE response pattern to increased O3 levels was a convex QE ( Figure 2C). In A. castellana WUE was higher in N and NFA+ than in CFA and NFA++ (QE t20 = −2.22 p = 0.04); a similar effect was observ in F. indigesta (t16 = −2.37, p = 0.03). Although in S. tenacissima the observed tre approached a convex response, the CE obtained the lowest p-value and was not signific castellana, S. tenacissima, F. indigesta, and F. iberica. Bars denote mean ± SE. Dotted lines denote marginally significant trends. Grey asterisk denotes marginally significant differences. Table S3 shows the lineal effect (LE), quadratic effect (QE), and cubic effect (CE) contrast for each species. Considering the O 3 effects on net photosynthesis (Figure 2A), a marginally significant LE was observed in A. castellana (t 20 = 1.93, p = 0.07). However, F. indigesta presented a marginally significant CE with maximum values under NFA+ (t 16 = −2.03, p = 0.06); a similar response was found in F. iberica although it was not significant (t 26 = −1.29, p = 0.21). The observed response of A in S. tenacissima was neither significant (t 30 = 1.61, p = 0.12 for a QE).

Gas Exchange
Regarding g s ( Figure 2B), three of the four species presented a similar concave trend in response to O 3 , with a greater stomatal opening under the lowest and highest O 3 concentrations (CFA and NFA++ treatments). In A. castellana, NFA and NFA+ treatments were marginally lower than CFA and NFA++ (t 20 = 1.79 p = 0.09); but even though S. tenacissima and F. indigesta showed the same concave pattern, they were not significant (t 30 = 1.42, p = 0.17; t 16 = 1.52, p = 0.15, respectively, for a QE). Contrary to conductance response in the other species, a marginally CE was observed in F. iberica (t 26 = −1.75, p = 0.09).
Consistently with g s values, the most observed WUE response pattern to the increased O 3 levels was a convex QE ( Figure 2C). In A. castellana WUE was higher in NFA and NFA+ than in CFA and NFA++ (QE t 20 = −2.22 p = 0.04); a similar effect was observed in F. indigesta (t 16 = −2.37, p = 0.03). Although in S. tenacissima the observed trend approached a convex response, the CE obtained the lowest p-value and was not significant (t 30 = −0.97, p = 0.34). As happened with g s , the WUE behavior in F. iberica was different from the rest of species with a significantly increasing LE (t 26 = 2.26, p = 0.03).
(F1, 9 = 3.59, p = 0.09). On the contrary, in the latter species, WUE was larger in NFA und the higher temperature values than in the AMB plots (F1, 9 = 5.65, p = 0.04). No differenc due to temperature were found for WUE in F. iberica (F1, 7 = 0.75, p = 0.41) and S. tenacissi (F1, 14 = 0.49, p = 0.49). The structure of the random part of the model for each variable a species is presented in the Table S5.  The temperature increase caused different effects on the gas exchange parameters depending on the species (Figure 3, Table S4). Photosynthetic activity was reduced in S. tenacissima plants grown inside the NFA OTCs compared with the AMB plots (F 1, 7 = 7.24, p = 0.02), but no temperature effect was detected in F. iberica (F 1, 7 = 1.73, p = 0.23) and A. castellana (F 1, 9 = 0.53, p = 0.48). Regarding g s , there was no difference between AMB and NFA in F. iberica (F 1, 7 = 1.75, p = 0.23) and S. tenaccissima (F 1, 14 = 0.17, p = 0.69). However, in A. castellana g s was marginally higher under the lower temperatures of the AMB plots (F 1, 9 = 3.59, p = 0.09). On the contrary, in the latter species, WUE was larger in NFA under the higher temperature values than in the AMB plots (F 1, 9 = 5.65, p = 0.04). No differences due to temperature were found for WUE in F. iberica (F 1, 7 = 0.75, p = 0.41) and S. tenacissima (F 1, 14 = 0.49, p = 0.49). The structure of the random part of the model for each variable and species is presented in the Table S5.

Nutrients and Nutrient Ratios
Nutrients (including C and H content) and nutrient ratios were differe spatially distributed among the treatments following MNDS (Non multidimensional scaling). MNDS results are presented for A. castellana and S. tena in Figure 4, and for both Festuca especies, F. indigesta and F. iberica, in Figure statistical differences among treatments that are shown below are based in PERMA analyses (Table S6). Regarding foliar nutrient content, A. castellana plants grown the lowest O3 exposure, the CFA treatment, had more Mn and P than plants grown the highest O3 exposure of the NFA++ (F3, 19 = 2.46, p = 0.02). In S. tenacissim concentration of S, Ca, Mg, Mn, B, and Zn was higher in CFA than NFA (F3, 19 = 2 0.03). PERMANOVA analysis also found significant differences among treatmen iberica (F3, 12 = 1.86, p = 0.048), with NFA having lower Fe, Mg, and Mo values tha Although NFA+ was also significantly different from NFA, the dispersion of th NFA+ points calls for caution in the interpretation of these results. However, ther no differences among treatments in F. indigesta for foliar nutrient concentration 0.81, p = 0.64). P/K and P/S nutrient ratios were different in CFA compared with the rest of treatments in A. castellana (F3, 19 = 3.02, p = 0.01). In S. tenacissima, only CFA and NF marginally different (F3, 19 = 2.06, p = 0.07) due to different P/S, N/K, P/K, Ca/M K/Ca, and K/Mg ratios. NFA had lower Ca/Mg and K/Mg ratios than NFA+ in F Bars denote mean ± SE. Temperature treatments: low temperature (AMB plots), high temperature (NFA OTC). Black and grey asterisk denote significant and marginally significant differences, respectively.

Nutrients and Nutrient Ratios
Nutrients (including C and H content) and nutrient ratios were differentially spatially distributed among the treatments following MNDS (Non-metric multidimensional scaling). MNDS results are presented for A. castellana and S. tenacissima in Figure 4, and for both Festuca especies, F. indigesta and F. iberica, in Figure 5. The statistical differences among treatments that are shown below are based in PERMANOVA analyses (Table S6). Regarding foliar nutrient content, A. castellana plants grown under the lowest O 3 exposure, the CFA treatment, had more Mn and P than plants grown under the highest O 3 exposure of the NFA++ (F 3, 19 = 2.46, p = 0.02). In S. tenacissima, the concentration of S, Ca, Mg, Mn, B, and Zn was higher in CFA than NFA (F 3, 19 = 2.34, p = 0.03). PERMANOVA analysis also found significant differences among treatments in F. iberica (F 3, 12 = 1.86, p = 0.048), with NFA having lower Fe, Mg, and Mo values than CFA. Although NFA+ was also significantly different from NFA, the dispersion of the three NFA+ points calls for caution in the interpretation of these results. However, there were no differences among treatments in F. indigesta for foliar nutrient concentration (  The lower temperature of AMB compared with NFA resulted in a higher P, Ca, Mg, and Fe content for A. castellana (F1, 9 = 3.68, p = 0.01). AMB had higher amounts of Mg, Ca, Mn, and Zn than NFA (F1, 10 = 5.15, p = 0.01) in S. tenacissima. Temperature caused no difference in nutrients content between AMB and NFA for either F. iberica (F1, 8 Figure 6 summarizes the significant correlations (p < 0.05) of nutrients content ( Figure  6A) and nutrient ratios ( Figure 6B) with gas exchange parameters (A, gs, and WUE) and aboveground vegetative biomass (leaf DW) for the four grasses studied. Photosynthetic P/K and P/S nutrient ratios were different in CFA compared with the rest of the O 3 treatments in A. castellana (F 3, 19 = 3.02, p = 0.01). In S. tenacissima, only CFA and NFA were marginally different (F 3, 19 = 2.06, p = 0.07) due to different P/S, N/K, P/K, Ca/Mg, C/N, K/Ca, and K/Mg ratios. NFA had lower Ca/Mg and K/Mg ratios than NFA+ in F. iberica (F 3, 12 = 2.8, p = 0.01). There were no differences among O 3 treatments in F. indigesta due to nutrient ratios (F 3, 14 = 0.7, p = 0.68).

Correlations
The lower temperature of AMB compared with NFA resulted in a higher P, Ca, Mg, and Fe content for A. castellana (F 1, 9 = 3.68, p = 0.01). AMB had higher amounts of Mg, Ca, Mn, and Zn than NFA (F 1, 10 = 5.15, p = 0.01) in S. tenacissima. Temperature caused no difference in nutrients content between AMB and NFA for either F. iberica (F 1, 8 = 2.31, p = 0.12) or F. indigesta (F 1, 9 = 1.68, p = 0.18). AMB plants presented higher ratios of P/K, P/S, and N/K and lower N/P than NFA plants in A. castellana (F 1, 9 = 5.53, p = 0.01). In S. tenacissima, AMB was significantly different from NFA due to different ratios of N/K, P/K, K/Ca, and K/Mg (F 1, 10 = 5.24, p = 0.02). There were no differences between AMB and NFA treatments in nutrients ratios in F. iberica (F 1, 8 = 1.68, p = 0.21) and F. indigesta (F 1, 9 = 1.57, p = 0.23). Figure 6 summarizes the significant correlations (p < 0.05) of nutrients content ( Figure 6A) and nutrient ratios ( Figure 6B) with gas exchange parameters (A, g s , and WUE) and aboveground vegetative biomass (leaf DW) for the four grasses studied. Photosynthetic activity was positively correlated with Mn and Mg, and g s increased with the amount of K. WUE was correlated positively with Zn, N/P, K/Ca, and K/Mg. On the other hand, higher K, Ca, Mg, Mn, and S were correlated with lower WUE values. Leaf DW was positively correlated with C/N, N/P, and K/Ca, but negatively with N, K, Ca, Cu, Fe, Mn, P, S, N/K, P/K, P/S, K/Mg, Ca/Mg.

Discussion
The present study reproduced the current and foreseen O3 levels and temperature increases on the pasture habitats of Mediterranean mountains. Ozone concentrations recorded at a mountain summit of the Spanish Central System yielded AOT40 values, cumulated over a 3-month period, ranging from 13,900 to 19,100 nL L −1 h depending among years [12]. These values greatly exceeded the current objectives for plant protection established in the EU directive of air quality (2008/50/EU), and the critical levels of the Air Convention for perennial pastures [9]. The equivalent estimated 3-month AOT40 index for NFA+ and NFA++ treatments in the present assay, comprising a 63-day fumigation period, would be about 12,000 and 22,000 nL L −1 h, respectively. Therefore, the O3 treatments in the experiment captured the interannual variability of O3 in the Iberian mountains. Furthermore, O3 concentrations in the NFA++ were also in the range of expected future O3 levels in Mediterranean mountain areas by 2050 [24].
Regarding the increase in temperature, previous studies have detected a temperature

Discussion
The present study reproduced the current and foreseen O 3 levels and temperature increases on the pasture habitats of Mediterranean mountains. Ozone concentrations recorded at a mountain summit of the Spanish Central System yielded AOT40 values, cumulated over a 3-month period, ranging from 13,900 to 19,100 nL L −1 h depending among years [12]. These values greatly exceeded the current objectives for plant protection established in the EU directive of air quality (2008/50/EU), and the critical levels of the Air Convention for perennial pastures [9]. The equivalent estimated 3-month AOT40 index for NFA+ and NFA++ treatments in the present assay, comprising a 63-day fumigation period, would be about 12,000 and 22,000 nL L −1 h, respectively. Therefore, the O 3 treatments in the experiment captured the interannual variability of O 3 in the Iberian mountains. Furthermore, O 3 concentrations in the NFA++ were also in the range of expected future O 3 levels in Mediterranean mountain areas by 2050 [24].
Regarding the increase in temperature, previous studies have detected a temperature increment in the Central System of 0.5 • C in the last decade, and projected temperatures in the Mediterranean mountains for 2085 could rise 5 degrees [50,51]. Therefore, the increase in temperature reached in the OTCs with respect to the AMB plots mimicked the expected increase caused by climate warming in the area. Nonetheless, the mean daylight temperatures in this study are within the range of the maximum values recorded in the natural environment of the species tested in the same year of the experiment (25.6 • C; [51,52]. The effect of the temperature increase found in this study could be slightly affected by the small differences between OTC and AMB in the other meteorological factors.
The O 3 and temperature sensitivity of the Mediterranean perennial grass species tested in this study does not present a single response or pattern. Alternatively, response patterns between species will be discussed in the light of their phylogenetic proximity and their adaptation to drought.

Effects on Plant Growth and Gas Exchange Parameters
As expected, the vegetative growth of Mediterranean perennial grasses showed a tolerant response to O 3 except in F. indigesta. This general lack of response to O 3 in growth parameters is consistent with previous results with grasses from natural ecosystems. Both Mediterranean annual and temperate perennial grasses have shown none or minor effects on growth under increasing O 3 exposure, as compared with O 3 sensitive legumes and forbs [31,33,34,41,42,53]. Despite this fairly widespread O 3 -tolerance of perennial grasses, some species such as Nardus stricta responded negatively to the pollutant [54]. In the present study, F. indigesta responded to the O 3 increase following a non-linear pattern: plants developed the highest biomass under the NFA and NFA++ treatments while minimum values were registered in the NFA+ with biomass reductions of 30% compared with CFA. F. indigesta showed a greater O 3 -sensitivity than its close phylogenic relative, F. iberica, despite the larger growth observed in the latter. F. iberica shows preference for wetter soils under natural conditions which would have been favored, as compared with F. indigesta, by the growing conditions in this study, where plants were kept with full water availability. A higher growth rate is usually associated with more intense physiological activity, greater gas exchange rates, and O 3 uptake [9]. Therefore, this study shows that the growth is not a reliable indicator of O 3 sensitivity for Festuca species. Interestingly, the O 3 levels in the natural mountain habitats of the Festuca species at the Spanish Central System during the spring season, when plants do not suffer drought stress and are fully physiologically active [12], are closely reproduced by the NFA+ conditions. Therefore, these results should warn of the current potential risk of O 3 negative effects in F. indigesta-dominated natural habitats, which are widespread in the Spanish Central System [28,55].
Only A. castellana responded positively to temperature increases. Considering the absence of soil moisture limitation in the present study, a higher growth under warmer conditions for all species could be expected [56]. During daylight hours, the mean temperature inside the OTCs was 26 • C during the experimental period, which is a common value for the natural habitat of this species in late spring and summer months. However, during the last 10 days of the fumigation experiment, temperatures rose inside the OTCs reaching values up to 40 • C. This late condition could hardly have affected the growth responses, since most of the growth occurred before the heat wave, and plants remained healthy without symptoms of heat stress. Therefore, A. castellana could potentially benefit, in the absence of water limitation, from the expected warming in its natural habitat in terms of aboveground biomass growth.
Gas exchange parameters were more sensitive to O 3 than biomass yield. F. iberica, F. indigesta, and A. castellana were affected differently according to the gas exchange parameter considered, while S. tenaccissima showed no effect. Previous studies have found that O 3 frequently inhibits A as a broad physiological response to the pollutant [57][58][59], response that has also been reported for grass species, and similarly this pollutant can also induce stomatal closure in O 3 -sensitive species [13,22,54,60,61]. In agreement with biomass growth responses, no species showed reductions of A in response to O 3 except F. indigesta and a marginal increase was even recorded in A. castellana. Both Festuca species showed a similar non-linear response pattern, but this effect was only marginally significant for F. indigesta. Strikingly, the highest A values measured in NFA+ were associated with the maximum growth loss in F. indigesta. A possible explanation is that photosynthate production under NFA+ was allocated to oxidative damage repair and defence [62][63][64][65], below ground biomass, or reproductive output [60,66]. However, similar A values in the NFA++ treatment would again be allocated to aboveground biomass growth. This kind of hormetic trend has been studied as an O 3 -triggered biphasic response under an oxidative stress [67]. A marginally linear positive response was found in A. castellana, not associated with higher aboveground biomass. In this case, like in F. indigesta, results suggest that higher amounts of photosyntates may have been allocated to other sinks under increasing O 3 stress.
The O 3 tolerance of plant species have been frequently related with stomatal behavior, since higher g s favors O 3 absorption and greater oxidative damage [68][69][70]. Of the species analyzed here, S. tenacissima and F. iberica would be the species with the maximum potential g s (based on CFA values). However, both species were tolerant in terms of growth and A. The O 3 -tolerance of the assayed grasses could then be related with a greater plant capacity for detoxification and repair, or a greater allocation to aboveground biomass, which has been considered among the main mechanisms underlying the tolerance of some species [60,66,71].
WUE was the gas exchange parameter most significantly affected by O 3 exposure in the present study. Ozone can unbalance WUE by inducing changes of different intensity in A and g s [72,73]. Ozone affected WUE in both Festuca species, although with different patterns. In F. iberica, the pollutant induced a reduction of stomatal opening maintaining A, resulting in a significant increase in WUE with increasing O 3 exposure. F. iberica can be classified as a "water use opportunistic" species [74], based on high g s and low WUE observed under the CFA treatment. However, under scenarios of high O 3 pollution, F. iberica would turn on a more "conservative" water use strategy. Therefore, the physiological response of F. iberica to O 3 levels expected in the future could help to simultaneously limit the effects of drought. Moreover, the WUE of this species was not affected by temperature. Therefore, the results would point towards a good performance of F. iberica under future combined scenarios of warming and O 3 pollution. F. indigesta showed the highest WUE under current O 3 levels in mountain areas (NFA+ treatment) of the species tested in this study, which is in concordance with its better adaptation to low soil water availability growing conditions [44]. However, increasing O 3 levels might affect the plant adaptation to these growing conditions, as O 3 induced a quadratic (hormetic) response in WUE, with the lowest WUE found under NFA++. The observed response of WUE to O 3 in A. castellana can also be explained as another hormetic response explained by effects on g s : first the rise of O 3 would induce a stomatal closure and then the excess of oxidative stress by higher O 3 levels would increase this parameter. This pattern of response could be related with a sluggish response of the stomata under O 3 stress [13,67,69]. The WUE response of A. castellana and F. indigesta pointed out in the same direction: both species maintained elevated A values under higher O 3 exposure, but at the cost of losing efficiency in the gas exchange process. Thus, the projected O 3 concentrations in mountain areas [24] could limit the tolerance of these species to the expected increases in drought [49] in these areas.
Contrarily to O 3 effects, the increase in temperature improved WUE rate in A. castellana because of the decrease in g s while maintaining A. The higher WUE was in agreement with a greater biomass growth. S. tenacissima, an O 3 tolerant species in terms of biomass growth and gas exchange, was the only species that lowered A values when grown at higher temperatures. S. tenacissima was sensitive to warming despite the fact that this species usually inhabits the driest and hottest environments of all the species tested [45,46]. Frequently, under well-watered conditions, a moderate increase in air temperature is accompanied by an increase in A [75,76]. However, the opposite response observed in S. tennaccissima could be more related with the hot temperatures reached at the late exposure period which can be affected the functioning of the photosystem II, accordingly with [77] for some herbaceous forbs.

Ozone and Temperature Effects on Foliage Nutrient Content
The sensitivity of foliar nutrient content and nutrient ratios to O 3 and temperature varied among the species and nutrients, or the nutrient ratios considered, in agreement with the results already published in the literature. A. castellana and S. tenacissima were the most sensitive species based on the number of affected nutrients (Figures S2 and S3), although O 3 sensitivity in S. tenacissima was based on the small increase in O 3 from CFA to NFA. A. castellana and S. tenacissima shared the same P/S, P/K, N/K, Ca, Mg, and Mn responses against the rise of O 3 and/or temperature. Among Festuca species, only F. iberica showed some response to O 3 in terms of nutrient content (Figures S4 and S5). Beyond the species-specific responses, the lack of similarity in the sensitivity of A. castellana and F. iberica to O 3 and temperature do not support the hypothesis that species sharing the same habitat would respond similarly to the abiotic factors tested in this study. On the other hand, the lack of effects on nutrient content and ratios in the Festuca species suggest that temperature sensitivity is phylogenetically related. Nevertheless, more studies involving more species are needed to unravel whether the nutrient response to O 3 and temperature is related to habitat adaptation or phylogeny.
Some of the responses found in the present work have been previously described for other species. Subtle effects of O 3 on the foliar C/N ratio in S. tenacissima and the absence of effects in the other species tested, contrast with O 3 -induced effects in foliar N observed in trees, pastures, and crop species, with both increasing and decreasing foliar N trends with O 3 exposure [16,78,79]. However, this result is in agreement with other studies reporting no effect in foliar N [80,81]. Beta vulgaris plants showed reductions in leaf Fe content in response to O 3 [18], like in F. iberica. Again, the O 3 effect on S. tenacissima promoting an increase in K/Ca, was also observed in the stomatal guard cells of the O 3 -injured leaves of Betula pendula [82]. Ozone induced decreases of foliar Mg and changes in the K/Mg ratio in F. iberica and S. tenacissima have also been described for Solanum tuberosum and Beta vulgaris [18,19,83]. Other studies, however, do not show O 3 effects on foliar nutrient concentration in species such as Lolium perenne or Trifolium repens, or a mixture of Trifolium pratense, Phleum pratense, and Festuca pratensis [80,84] Temperature effects on foliar P and Fe in A. castellana have also been described in Coffea canephora and in plants from a dwarf shrub-dominated ecosystem [85,86]. However, the negative temperature effect on Mg in A. castellana was not found in Mediterranean scrub [87]. These results show that O 3 and temperature effects on foliar nutrient content and ratios seem to be species-specific and variable between nutrients. More studies would be needed for understanding general response patterns.
Nutrient and nutrient ratios were more correlated with vegetative growth than with gas exchange parameters. The influence of O 3 and temperature on leaf nutrient concentration and the relationship with vegetative growth, A, g s , and WUE constitute a complex web of interactions (Figures S2-S5). Considering all four species together, the vegetative growth was negatively correlated with most of the macro-and micronutrients and the different ratios considered (Figure 6). The dilution of nutrient content related with biomass growth has been shown for N, P, and K [88,89]. These nutrients are directly or indirectly related to plant development [90,91]. In general, N-and P-deficiency inhibits leaf growth and Fe is mostly located in the chloroplast of growing leaves [90,91]. In A. castellana, the temperature effect on leaf nutrients could be explained by a dilution effect due to increases in vegetative growth under the warmest treatment. The N/P ratio was also positively correlated with vegetative growth in this species, which is in agreement with temperature-induced increases in this ratio. None of effects of O 3 on foliar nutrient content could be related with effects on biomass growth in any of the four species. However, O 3 effects on foliar nutrient contents in A. castellana, F. iberica, or S. tenacissima found in this short-term exposure study may indicate that effects on plant growth could appear under long-term high O 3 exposures.
In the present study, Mn and Mg were positively correlated with A. In fact, Mn and Mg act synergistically in the basal metabolism of electron transport reactions in photosynthesis [92,93]. An ozone-induced decrease in foliar Mg has been related with chlorophyll loss and A decline [18,19,83]. In contrast to this, O 3 exposure increased A in A. castellana associated with a decrease in leaf Mn content ( Figure S2). However, in S. tenacissima the decrease in Mn and Mg under the high temperature treatment could be associated with the decrease in A ( Figure S2). Foliar K was positively correlated with g s across species. This correlation is in accordance with the stomatal hydration regulatory function of K that produce the stomata opening due to increased turgor in guard cells [90,92]. Despite O 3 and temperature effects found on the g s of A. castellana and F. iberica, leaf K content was not affected by the experimental treatments in any of the four species studied (Figures S2 and S5). Previous studies neither found significant effects on the K-levels of potato or wheat exposed to increased levels of O 3 [19,94]. S, Ca, K, Mg, and Mn foliar concentrations were negatively correlated with WUE while Zn and N/P, K/Ca, and Ca/Mg ratios were positively correlated ( Figure 6). These relationships can be explained through the importance of some of these elements on A or g s processes. Stomatal opening by K accumulation is promoted by Ca signaling [90][91][92] which could result in decreased WUE. Zn-mediated stability of membrane integrity [91,93] may explain the higher WUE under high foliar Zn levels while decreases in foliar Mg could reduce WUE via reductions in A. The O 3 -induced decreases in Mn and P and in the P/K and P/S ratios in A. castellana can be associated directly or indirectly with the observed quadratic effect on WUE. However, the quadratic effect of O 3 on WUE in F. indigesta was not associated with any significant change in foliar nutrient concentration. With respect to the temperature effects in A. castellana, the increase observed in WUE was associated with decreases in Ca and Mg concentration and a reduction in the N/P ratio.

OTC Experiment
Plant exposure to O 3 treatment levels was performed in an NCLAN-type OTC facility (adapted from the original design by [95]) located in central Spain (450 m.a.s.l., 40 • 3 N, 4 • 26 W). Plants were exposed to four O 3 treatments: charcoal-filtered air (CFA), non-filtered air (NFA) reproducing ambient levels, non-filtered air supplemented with 20 nL L −1 of O 3 (NFA+), and non-filtered air supplemented with 40 nL L −1 of O 3 (NFA++). Each O 3 treatment was replicated 3 times in 12 equally built OTCs randomly distributed in 3 lines (blocks) avoiding shading effects between OTCs. One chamber-less plot (AMB) per block was considered to control the chamber effect and to study the consequences of temperature increase on species.
Ozone was produced from pure O 2 through an O 3 generator (A2Z Ozone, Inc., Louisville, KY, USA) and supplied to the NFA+ and NFA++ plots 8 h day −1 (7:00 to 15:00 GTM) and 7 days week −1 . Ozone concentrations inside each chamber and AMB plots were monitored continuously above plant canopy using a UV-absorbance O 3 monitor (ML ® 9810B, Teledyne, Thousand Oaks, CA, USA) with an automated time-sharing system sampling all the OTCs and AMB plots of each line sequentially. Another O 3 monitor within the fumigation system registered continually ambient O 3 levels to contrast with the NFA and AMB treatments, as a double check of the accuracy of O 3 values. Both O 3 monitors were calibrated at the start of the fumigation treatments following the recommended company protocols. More information about the facility can be found in [22]. Figure S1 shows a picture of the OTCs and AMB plots.

Plant Material
Established seedlings of A. castellana, S. tenacissima, F. indigesta, and F. iberica grown from seeds collected from natural populations of Spanish Central System area were transplanted to 2 L pots during 2017 spring. A mix of peat, vermiculite, and perlite (60:20:20) was used as plant substrate. The total number of plants varied among species, with 90, 88, 77, and 51 for A. castellana, S. tenacissima, F. iberica, and F. indigesta, respectively. Plants were kept outdoors since transplantation. They were frequently irrigated and fertilized to meet plant demand, until the start of the experiment. On 15 April 2018, 3 days prior to the start of the O 3 -exposure, plants were cut to 5 cm from the substrate. These species, adapted to cattle browsing, present a good regrowth after biomass cut. On 18 April, plants were randomly allocated to OTCs or AMB plots to start the O 3 fumigation experiment. The 4 species were exposed to the same O 3 -treatments that lasted 68, 63, 62, and 57 days after the start of the exposure (DaS) for A. castellana, S. tenacissima, F. indigesta, and F. iberica, respectively.

Vegetative Growth
At the end of the O 3 -fumigation period, plants were cut again to 5 cm from the substrate to obtain the final aboveground biomass. Samples were dried at 60 • C until constant weight. The O 3 effect on the dry weight biomass (biomass DW, g) was considered to discuss the O 3 -effect on the vegetative growth of the species. The number of samples per treatment and species are shown in Table S1.

Gas Exchange
Leaf-level net photosynthesis (µmol CO 2 m −2 s −1 ) and stomatal conductance to water vapor (mol H 2 O m −2 s −1 ) were measured using a portable LICOR-6400 infra-red gas analysis system (LiCor Inc., Lincoln, NE, USA). The Water Use Efficiency (WUE; µmol CO 2 mol −1 H 2 O) was calculated from the A/g s ratio. In order to increase the leaf area for the gas exchange measurements, a group of intact leaves was introduced in the LICOR chamber. Each individual measurement was corrected per projected leaf area based on the scanned image of leaf sections measured with ImageJ software [97]. The number of samples per treatment and species are shown in the Table S1. Gas exchange measurements were evenly distributed between 9 and 13 h (GMT) among all treatments, from 28 to 35 days after the start of the fumigation date depending on the phenological stage of each species (Table S2). Air humidity and temperature during the measurements ranged 36-49% and 20-25 • C, respectively, and PAR was maintained at 1000 µmol m −2 s −1 , allowing maximum g s according to previous measurements (Table S2). One measurement per plant was taken on the representative biomass of the plant, avoiding the youngest leaves in growth and the oldest senescent leaves. The arrival of a heat wave towards the end of the ozone fumigation experiment prevented the measurement of gas exchange on ambient F. indigesta plants.

Macro-and Micronutrient Composition
Total aerial biomass DW from the different plants was individually milled and pooled to obtain a minimum of 2 independent samples (no plant was repeated in the mixtures) per OTC and AMB plot for elemental content analysis. The number of samples per treatment and species are shown in Table S1. Total carbon and hydrogen content (C and H), macronutrients (N, K, Ca, Mg, P, and S) and micro-nutrients (B, Cu, Fe, Mn, Mo, and Zn) were analyzed and the ratios C/N, N/K, N/P, P/K, P/S, K/Ca, K/Mg, and Ca/Mg were also calculated. The total content of C, H, and N were determined by combustion using an elemental analyzer. Samples were digested under controlled conditions for putting quantitatively into solution these macronutrients (K, Ca, Mg, P, and S) and micronutrients (B, Cu, Fe, Mn, Mo, and Zn). Concentrations of these elements were determined by inductively coupled plasma optical emission spectrometry (ICP-OES). More details on sample preparation and elemental analyses are provided in the suplementary information.

Statistical Analysis
To analyze the effect of O 3 and temperature factors separately on vegetative growth, A, g s , and WUE, two sets of linear mixed models (LMM) were performed. The first set of LMM considered the O 3 treatments (CFA, NFA, NFA+, and NFA++). In the second set, the comparison between AMB and NFA was considered to study the effect of temperature. For each species, the individual plants were the units of replication. Moreover, to control the possible influence of the experimental design in plants growth, A, g s , and WUE responses to the fixed factors (O 3 and temperature), block (line), and O 3 treatment nested within line were considered as random factors. For each analysis, the AIC (Akaike information criterion) value was used to choose the most parsimonious option between the models including only line as a random factor or the nested model. Linear O 3 effects were evaluated using linear a priori contrasts to test the stated hypothesis. Moreover, quadratic and cubic responses were also tested. A priori contrasts in linear mixed models were based on [98]. Linear mixed models were performed using lme function (nlme package; [99]). To test the hypotheses of a linear, quadratic, or cubic effect the contr.poly function [100] was used. The model with the lowest p-value was chosen among lineal, quadratic, and cubic options for each analysis. Temperature effects were tested using ANOVA function.
To evaluate the O 3 and temperature effect on leaf nutrient composition, PERMANOVA and Non-Metric Multidimensional Scaling (NMDS) analyses were performed using leaf nutrient concentration and nutrient ratios as a multivariate trait among treatments. To prevent PERMANOVA from being largely influenced by the high levels of some elements, the data were transformed using the square root, and a similarity matrix was made by Bray-Curtis approximation [101]. A total of 9999 permutations were chosen, and PERMANOVA was performed using vegan package (Oksanen et al., 2020). Pairwise analyses were performed when PERMANOVA found differences among O 3 -treatment using pairwise.adonis2 function (package pairwiseAdonis; [102]). Two-dimension NMDS were used to represent the elemental composition multidimensional data. Function metaMDS was used to perform the NMDS (vegan library; [103]). Correlations of nutrients and nutrient ratios with vegetative growth, A, g s , and WUE were also performed using vegan package [103]. All the statistical analyses described were carried out in R software [100]. Significant statistical differences were considered at p-values lower than 0.05. Marginally statistical differences were considered at p-values from 0.1 to 0.05.

Conclusions
Gas exchange parameters, in particular WUE, and leaf nutrient concentration were more sensitive to O 3 and temperature increases than aboveground biomass growth in the Mediterranean perennial grass species assayed in this experiment.
The O 3 sensitivity classification of grasses based on growth and gas exchange does not match with that based on foliar nutrients. S. tenaccissima was the most tolerant to O 3 in terms of growth and gas exchange. However, A. castellana were the most sensitive to the pollutant in terms of nutrients. In both Festucas, gas exchange, growth, and leaf nutrient content had different responses to O 3 . The O 3 -effects on foliar nutrients and their ratios, although substantial, did not have an early impact on plant growth. Longer term exposures will be needed to understand the potential consequences of these changes for the growth and survival of these species.
S. tenacissima and A. castellana were the two most sensitive grasses to temperature increase. S. tenacissima was the only one that decreased A at high temperatures, despite the fact that it usually inhabits the driest and hottest environments of all the species tested. A. castellana was the only species that responded positively in terms of aboveground biomass and WUE when grown in a warmer environment. This species could benefit from climate warming when water is not a limiting factor. However, it was the most temperaturesensitive in terms of foliar nutrients, so the long-term effects may differ from the ones found in this experiment. Only leaf nutrient content responses to temperature seem to be phylogenetically constrained in Festuca species.
The results showed that responses to O 3 and temperature can have different speciesspecific effects on plant physiology, potentially altering the ability of plants to cope with environmental stresses and changing the relationships among species that share the same habitat. Therefore, climate warming and O 3 pollution should be considered as two important threats to Mediterranean perennial pastures in the framework of the Global Change. More experimental work would be needed to better understand the behaviour of these complex Mediterranean pastures in response to combinations of stress factors.

Supplementary Materials:
The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/plants12030664/s1, Figure S1: Picture of two OTCs and one AMB plot of the OTC facility; Description of the description of the total content of C, H, macro-and micro-nuctrient determitaion; Table S1: Number of samples per treatment and specie for Vegetative growth (dry weight), Gas Exchange (A, g s and WUE) and Nutrient (contents and rates) measurements; Table S2. LICOR measurements conditions for photosynthesis and conductance; Table S3: A priori contrast results of the lineal effect, quadratic effect and cubic effect for each specie in vegetative growth, A, g s and WUE; Table S4: Temperature effect results on vegetative growth, A, g s and WUE; Table S5: The structure of the random part of the model for each trait (vegetative growth, photosynthesis, conductance and WUE) and species; Table S6: PERMANOVA tables of nutrient and nutrients ratios for O 3 and temperature; Figure S2: Ozone and temperature observed effects on growth and gas exchange measured traits and nutrients in A. castellana; Figure S3: Ozone and temperature observed effects on growth and gas exchange measured traits and nutrients in S. tenacissima; Figure S4: Ozone and temperature observed effects on growth and gas exchange measured traits and nutrients in F. indigesta; Figure S5: Ozone and temperature observed effects on growth and gas exchange measured traits and nutrients in F. iberica. Data Availability Statement: Publicly available datasets were analyzed in this study. This data can be found here: http://rdgroups.ciemat.es/web/geca-ciemat/ (accessed on 20 January 2023).